In a recent study published in Emerging Infectious Diseases, researchers conducted bat sarbecovirus isolation experiments using Rhinolophus cornutus bats from various regions of Japan that were phylogenetically in the same group of viruses related to respiratory syndrome coronavirus 2 severe acute (SARS-CoV-2). .
Study: Isolation of Bat Sarbecovirus, Japan. Image credit: Independent Birds/Shutterstock
Highly pathogenic human beta-coronaviruses (β-CoVs) such as severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome CoV have been considered (MERS-CoV) have originated from bats. Therefore, bat β-CoV surveillance is essential to improve understanding and to assess the potential for β-CoV spillovers in humans. Sarbecoviruses identified in Asian nations such as China have been reported to be genetically diverse; however, the genetic variations and distribution of bat sarbecoviruses in Japan have not been well characterized and require further research.
The authors of the present study previously showed that a VSV (vesicular stomatitis virus)-based viral pseudotype that includes the S (spike) protein of the R. cornutus sarbecovirus Rc-o319 infected only Rc-ACE2 (converting enzyme angiotensin 2) expressing cells, but not human ACE2-expressing cells (hACE2) or other Rhinolophus ACE2-expressing cells.
About the study
In the present study, researchers reported the identification, isolation, and biological and genetic characterization of sarbecoviruses originating from bats in different Japanese locations.
Faecal samples were obtained from the bat species R. ferrumequinum and R. cornutus in Chiba, Shizuoka, and Niigata prefectures. Real-time RT-PCR (reverse transcription polymerase chain reaction) was performed, and the team established Vero cells expressing RcACE2 (Vero-RcACE2) based on Vero cells/transmembrane serine protease 2 ( TMPRSS2).
Next-generation sequencing (NGS) analysis was performed to determine the whole-genome sequence of all viral isolates, and the sequences were deposited in GenBank. In addition, a similarity plot analysis was performed for the complete genome sequence using each isolate as a query. A phylogenetic tree of bat sarbecoviruses in Japan was constructed based on whole genome sequences with maximum likelihood analysis and bootstrapped replicates.
The S RBM (receptor binding motif) of bat isolates from Japan was aligned with those of other sarbecoviruses. To evaluate the growth kinetics of bat sarbecovirus isolates in Japan, R. cornutus bat isolates Rc-os20, Rc-o319, Rc-mk2, and Rc-kw8 or SARS-CoV-2 (strain B .1.1.7). in Vero-RcACE2 [RcACE2, Vero/TMPRSS2 (WT)]Vero-hACE2 (hACE2) or Vero-ACE2KO (ACE2KO) and viral titers were determined by plaque assays.
results
The isolates showed efficient growth in cells expressing Rhinolophus cornutus ACE2, but did not grow well in cells expressing hACE2, indicating that the bat isolates had a narrow host range. RT-PCR analysis allowed the successful detection of the envelope (E) gene sequence of sarbecovirus in one or two samples of Rhinolophus cornutus in each prefecture. Bat sarbecoviruses were successfully isolated using Vero-RcACE2 cell cultures, with huge cytopathic effects and syncytium formation from RT-PCR positive samples of bat feces from all prefectures.
Isolates from bats from Shizuoka, Niigata, and Chiba prefectures were designated as Rc-kw8, Rc-os20, and Rc-mk2, respectively. In addition, Rc-o319 was isolated using Vero-RcACE2 cell cultures. In contrast, all samples of Rhinolophus ferremuquinum species were negative, indicating that bat sarbecoviruses were distributed among Rhinolophus cornutus bats in Japan. The sequences of all bat strains isolated from Japan were highly homologous (ranging from 95% to 97%). However, Rc-os20 and Rc-mk2 lacked the region coding for ORF8 (open reading frame 8).
The team observed high similarities between the bat isolates throughout the genome sequence, with the exception of the sites encoding the gene S receptor binding domain (RBD) and the N-terminal domain (NTD) . However, NTDs Rc-kw8 and Rc-o319 were found to be conserved. No significant recombination was observed among the bat isolates. In the phylogenetic analysis, the isolates were observed in a genetic cluster located in a genetic clade that includes sarbecoviruses related to SARS-CoV-2.
The results of the S RBD phylogenetic analysis indicated that the isolates from Japan were positioned in the genetic clade of viral organisms that use orthologous ACE2 molecules as receptors. Therefore, the team assumed that the new strains isolated from Japan use RcACE2 as receptors. The bat isolates replicated efficiently only in Vero-RcACE2 but not in Vero-ACE2KO, Vero/TMPRSS2, or Vero-hACE2 cells. The findings indicated that the bat isolates depended on RcACE2 for their infectivity.
In contrast, SARS-CoV-2 showed efficient replication in Vero-RcACE2, Vero-hACE2, and Vero/TMPRSS2 cells, but did not replicate well in Vero-ACE2KO cell cultures, indicating that the its infectivity depended on the presence of multiple ACE2 receptors. including those of Rhinolophus cornutus bats. The findings indicated that bat isolates from Japan used only bRcACE2 as receptors.
Most of the genome sequences among the strains from Japan were highly conserved, which could be from Rhinolophus spp. bat species migrate relatively short distances and do not usually cross-contact with other bat groups. The exceptions were the S regions encoding RBDs and NTDs, which showed high genetic variation due to immunological pressures, indicating that bats diverged from the ancestral strain in recent times.
Overall, the findings of the study showed that the bat isolates used batACE2 in the form of non-replicating receptors in hACE2-expressing cells, thus being a characteristic type and indicating a low potential for human infection. Sarbecoviruses can mutate and cause human infections through intermediate host species in livestock or wildlife. Therefore, epidemiology studies of sarbecoviruses in wild animals, including bats, with long-term risk assessments of their potential for zoonotic transmission should be conducted.